Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes

Proteinuria in Lamb2^−/−; NEPH-C321R-LAMB2 mutant mice correlates inversely with the level of transgene expression before the first month of age. Urinary protein/creatinine ratios (gram/gram) between 3 and 4 weeks of age in the three lines of Lamb2^−/−; C321R-LAMB2 mice and their WT and nontransgenic Lamb2^−/− littermates are shown in the graph as mean ± SD. **P<0.001. NS, not significant. Statistical analysis was done by ANOVA with the Tukey multiple comparison test.

Ectopic deposition of laminins α1, α2, and β1 in the GBM is associated with reduced expression of β2 in the GBM of Lamb2^−/−; C321R-LAMB2 mice. Frozen kidney sections from Tg^Hi mutants, their WT and nontransgenic Lamb2^−/− littermates, and age-matched Lamb2^−/−; Tg-WT mice were stained for laminin chains at 3 weeks. (A and E) The normally mesangial laminins α1 and α2 were ectopically deposited in the (B and F, arrows) Tg^Hi and (C and G, arrows) Lamb2^−/− GBM but not (D and H) Tg-WT GBM. I–L show merged images for which overlapping laminin β1 (red) and nidogen (green) signals appear (J and K, arrows) yellow in the GBM of Lamb2^−/−; Tg^Hi and Lamb2^−/− mice as well as (I–L) the mesangium of all mice, where both proteins are normally present. Additional data are shown in Supplemental Figure 1. Scale bar, 20 µm.

Light and electron microscopy analyses of kidneys from the three lines of Lamb2^−/−; NEPH-C321R-LAMB2 mice and their Lamb2^−/− and WT littermates at 3 weeks show that glomerulosclerosis and mild GBM thickening in Lamb2^−/− mice are attenuated by increased expression of C321R-LAMB2 in the GBM. (A) Paraffin sections of WT and mutant kidneys were stained with H&E (HE) and PAS. Lamb2^−/− mice showed (d) glomerulosclerosis, (h, arrow) prominent renal tubular protein casts, and (h) loss of brush borders compared with (a and e) the WT littermates. Tg^Lo Lamb2^−/−; C321R-LAMB2 mice exhibited (b) MM expansion, (f, arrow) fewer protein casts, and (f) partial loss of brush borders. (c and g) No obvious pathology was detected in Tg^Hi mutants. Scale bars, 20 µm in a–d; 100 µm in e–h. (B) TEM showed diffuse FP effacement (*) and mild GBM thickening (arrow) in (e) Lamb2^−/− mice compared with (a) controls. FP effacement (*) was observed in (b–d) all mutant lines, with some GBM outpocketing in the (b, arrows) Tg^Lo and (c, arrows) Tg^Med mutants. Insets in b and c magnify regions between the two arrows. (f–h) Focal areas with intact foot processes were also found in the three mutant Tg lines. Scale bar, 500 nm.

Increased level of C321R-LAMB2 in the GBM is associated with less interstitial fibrosis at 6 weeks. TEM analysis showed irregular GBM thickening (arrows) and diffuse FP effacement (*) in Tg^Lo, Tg^Med, and Tg^Hi Lamb2^−/−; C321R-LAMB2 mice. Scale bar, 500 nm. Gomori’s Trichrome staining revealed less interstitial fibrosis in Tg^Med and Tg^Hi mutants compared with Tg^Lo mutants (arrows). Scale bar, 100 µm.

Secretion of the mutant laminin β2 fragment/Gluc fusion protein is inhibited and podocyte ER stress is induced in Tg^Hi Lamb2^−/−; C321R-LAMB2 mice. (A, arrow) The WT but not the C321R-LAMB2/Gluc fusion protein was secreted from stably transfected HEK293T cells. (B) This finding was reflected by luciferase activity in the media (**P<0.001 by t test). The data are presented as mean ± SD of three independent media samples. (C) The C321R/Gluc fusion protein was retained intracellularly compared with the WT fusion protein. (D) Quantitative RT-PCR did not show a significant difference in Lamb2 mRNA levels between the WT and C321R cell clones. Data are presented as mean ± SD of fold changes of four independent samples from each clone. P>0.05 by t test. (E) Colocalization of the (a) WT/Gluc or (b) C321R LAMB2/Gluc fusion protein with the (c and d) ER marker PDIA3 in the 293T-Gluc cells using confocal microscopy. (e and f) Nuclei were counterstained with Hoechst 33342 (blue). (a, c, and e) The WT fusion protein seemed to be transiently associated with PDIA3, but (b, d, and f) the mutant was completely colocalized with PDIA3, indicating accumulation in the ER. (c and d) The mutant protein also induced expression of PDIA3. Scale bar, 10 µm. (F) Cell lysates of 293T-Gluc clones were subjected to Western blot analysis. The C321R but not the WT fusion protein increased BiP expression (arrow). Tunicamycin (TM) -treated cells served as positive controls. (G) Frozen kidney sections from mice of the indicated genotypes were examined by dual immunofluorescence staining of BiP and WT-1 at 3 weeks. Compared with the very low level expression of (a, e, and i) BiP in the control podocytes, BiP upregulation was detected in podocytes of (c, g, and k) Tg^Hi glomeruli and (d, h, and l) some Lamb2^−/− glomeruli but not (b, f, and j) podocytes of Tg-WT glomeruli. Scale bar, 20 µm.

C321R-LAMB2 causes podocyte ER distention, upregulation of CHOP, and activation of desmin in podocytes at 3 weeks. (A) TEM showed that rER in the podocytes of (b, arrows) Tg^Hi Lamb2^−/−; C321R-LAMB2 and (c, arrows) Lamb2^−/− mice was dilated compared with rER in the (a) podocytes of age-matched Lamb2^−/−; Tg-WT controls. The swollen, ribosome-studded (b, arrows) tubular structures or (c, arrows) vesicles identify the rER. Scale bar, 500 nm. (B) Double immunofluorescence staining for (a–c) CHOP and (d–f) WT-1 on frozen kidney sections revealed podocyte induction of CHOP in (b, e, and h) Lamb2^−/−; Tg^Hi and (c, f, and i) Lamb2^−/− glomeruli but not (a, d, and g) Tg-WT glomeruli. Scale bar, 20 µm. (C) Frozen sections of Lamb2^−/−; Tg-WT, Lamb2^−/−; Tg^Hi, and Lamb2^−/− kidneys were stained for (a–c) podocin and (d–f) desmin and (g–i) merged. Compared with (a, d, and g) controls, desmin expression was activated in the podocytes of (b, e, and h, arrows) mildly proteinuric Tg^Hi mutants and (c, f, and i, arrows) highly proteinuric Lamb2^−/− mutants. Scale bar, 20 µm.